Type I diabetes, or IDDM, is an autoimmune disease caused by T cells that attack and destroy the insulin-producing .beta.-cells located in the islets of the pancreas (Castano and Eisenbarth, 1990). The autoimmune process culminating in IDDM begins and progresses without symptoms. The disease surfaces clinically only when the cumulative loss of .beta.-cells exceeds the capacity of the residual .beta.-cells to supply insulin. Indeed, the collapse of glucose homeostasis and clinical IDDM is thought to occur only after 80-90% of the .beta.-cells have been inactivated by the immune system. Thus, patients who can be identified as suffering from IDDM are bound to be in an advanced stage of autoimmune destruction of their .beta.-cells. Moreover, diagnosis of incipient, pre-clinical diabetes by the detection of immunological markers of .beta.-cell autoimmunity can be made only after the onset of the autoimmune process. Therefore, the therapeutic quest is to find a safe, specific and effective way to turn off an autoimmune process that is already well underway.
The present inventors have examined this question before by studying the spontaneous diabetes developing in mice of the NOD strain, which is considered to be a faithful model of human IDDM (Castano and Eisenbarth, 1990). NOD mice develop insulitis around 4 weeks of age, which begins as a mild peri-islet infiltrate and progresses to severe intra-islet inflammation. Hyperglycemia, which attests to insulin insufficiency, begins in the females in our colony at about 14-17 weeks of age. By 35-40 weeks of age, almost all the female NOD mice have developed severe diabetes and most die in the absence of insulin treatment. Male NOD mice have a lower incidence of disease, for reasons that are not clear. The diabetes of NOD mice has been shown to be caused by autoimmune T cells (Bendelac et al., 1987).
T cell reactivity and autoantibodies to various antigens have been detected in human IDDM patients as well as in NOD mice (Elias, 1994), and it is not clear whether immunity to any single one of the possible target antigens is the primary cause of the disease. Beyond the question of causation is the question of therapy.
It has been demonstrated that the initiation of the autoimmune process in NOD mice can be prevented by subjecting the mice, before the onset of diabetes, to various manipulations such as restricted diet, viral infections, or non-specific stimulation of the immune system (Bowman et al., 1994). NOD diabetes is also preventable by induction of immunological tolerance in pre-diabetic mice to the antigen glutamic acid decarboxylase (Kaufman et al., 1993; Tisch et al., 1993).
Insulin dependent diabetes mellitus (IDDM) developing spontaneously in NOD female mice has been associated with immune reactivity to a variety of self-antigens (Bach, 1994). Notable among these antigens is the p277 peptide from the sequence of the mammalian 60 kDa heat shock protein (hsp60) molecule. This corresponds to residues 437-460 in the human hsp60 molecule (Elias et al 1991, Israel Patent Application No. 94241, PCT patent publication W090/10449). The human p277 peptide has the following sequence:
Val-Leu-Gly-Gly-Gly-Cys-Ala-Leu-Leu-Arg-Cys-Ile-Pro-Ala-Leu-Asp-Ser-Leu-Thr -Pro-Ala-Asn-Glu-Asp (a.a. 437-460 of SEQ ID NO:1). PA1 (i) preparing a mononuclear cell fraction containing T cells from a blood sample obtained from said patient; PA1 (ii) adding to said mononuclear cell fraction an antigen selected from the peptide of the invention; PA1 (iii) incubating said cell fraction in the presence of said antigen for a suitable period of time and under suitable culture conditions; PA1 (iv) adding a labeled nucleotide to the incubated cell culture of (iii) at a suitable time before the end of said incubation period to provide for the incorporation of said labeled nucleotide into the DNA of proliferating T cells; and PA1 (v) determining the amount of proliferating T cells by analysis of the amount of labeled nucleotide incorporated into said T cells. PA1 In step (iv) above, said labeled nucleotide is preferably 3H-thymidine. The determination of the amount of proliferating T cells is made by calculation of the stimulation index of the T cells by standard methods. PA1 (i) an antigen selected from the peptides of the invention; and PA1 (ii) a tagged antibody capable of recognizing the non-variable region of said anti-hsp60 antibodies to be detected. PA1 A kit for diagnosing the presence of IDDM by testing for the presence of a T cell which immunoreacts with hsp60, will comprise: PA1 (i) an antigen selected from the peptides of the invention; PA1 (ii) a suitable medium for culture of lymphocytes (T cells); and PA1 (iii) either a labeled nucleotide for the T cell proliferation test, or a cytokine, e.g., interferon-gamma, assay kit, for the cytokine test.
Pre-diabetic NOD mice manifest spontaneous, diabetogenic T cell responses to hsp60 and to the human (2) or mouse variants of the p277 peptide (3). The mouse and human peptides differ by 1 amino acid and are immunologicaly cross-reactive (3). Some non-diabetes prone strains of mice, such as C57BL/6, develop transient hyperglycemia and insulitis when immunized to p277 covalently conjugated to a foreign immunogenic carrier molecule (4). And mice of the C57BL/KsJ strain develop spontaneous T-cell responses to hsp60 and to p277 after treatment with a very low dose of the .beta.-cell toxin streptozotocin (STZ) that induces autoimmune diabetes (5).
In addition to being involved in the expression of the disease, peptide p277 appears to be functional in healing the autoimmune process: Subcutaneous administration of p277 in incomplete Freund adjuvant (IFA; mineral oil) led to arrest of disease progression in young NOD mice (2) or in 12-17 week old NOD mice with advanced insulitis (6, 7). Both the human (6, 7) and mouse (3) variants of p277 were effective. NOD mice transgenic for the mouse hsp60 gene on an MHC class II promoter showed down-regulation of their spontaneous T-cell proliferative response to p277 and a significant proportion of the mice were spared the development of diabetes (8). Moreover, administration of p277 to C57BL/KsJ mice aborted the development of autoimmune diabetes in mice that had received earlier a very low dose of STZ; treatment of these mice with a peptide of the GAD65 molecule was not effective (9).
Variants of the p277 peptide in which one or both cysteine residues at positions 6 and 11 were replaced by valine residues, designated as p277(Val.sup.6), p277(Val.sup.11) and p277(Val.sup.6 -Val.sup.11), respectively, were described in corresponding Israel Patent Application No. 112094, and shown to be as active as p277 in the treatment of diabetes.
It is an object of the present invention to provide additional peptides of human hsp60, such peptides being useful for diagnosis and treatment of IDDM.